Phospholipids in biological membranes are asymmetrically oriented across the bilayer, the choline containing phospholipids are localized primarily in the membrane outer monolayer and the amine-containing phospholipids, phosphatidylethanolamine and phosphatidylserine (PS), are sequestered in the membrane inner monolayer. Loss of PS asymmetry is associated with cell senescence and contributes to thromboses in heart disease, stroke, and diabetes. Phospholipid asymmetry in maintained in part by ATP-dependent transport of aminophospholipids from the membrane outer to inner surface. Aminophospholipid transport, or "flippase", activity has been well characterized in the plasma membrane and internal organelles of a variety of cells. Transport requires Mg2+-ATP and is inhibited by vanadate, sulfhydryl or arginine modification, and calcium. Recently, two proteins have been proposed as candidate transporters: a vanadate-sensitive PS-dependent ATPase (about 120 Kda) and a protein that reacts with sulfhydryl and PS affinity probes (32 Kda). A preliminary purification of the vanadate-sensitive, PS-dependent ATPase suggests that the flippase may be a complex of several proteins. The goal of the proposed work is to identify, purify and reconstitute the aminophospholipid flippase. Three specific aims will be addressed: 1) Improved sulfhydryl and photoaffinity (carbene and radical) PS analogs will be synthesized. These affinity lipids will be used to identify PS-binding and sulfhydryl-containing proteins in human erythrocytes. 2) Candidate aminophospholipid transporters will be purified from erythrocytes and rat brain using standard chromatographic and novel affinity methods. A unique PS affinity matrix will be constructed to selectively purify PS-binding proteins. The partially purified ATPase will be fractionated by gel-filtration and additional affinity purification methods. Distinguishing characteristics of PS transport, including PS and ATP binding, will be determined. Antibodies will be raised against purified proteins and will be used as probes of the structure, function, identification, and eventual molecular cloning of the flippase. 3) Purified candidate transporters will be reconstituted into liposomes and ATP-dependent transport activity will be measured. Flippase activity will be determined by measuring changes in PS transmembrane distribution with an enzymatic assay based on the PS-dependent activation of protein kinase C and by fluorescence resonance energy transfer between labeled phospholipids. The proposed studies may result in the identification and functional reconstitution of the aminophospholipid flippase. Future molecular biological and biophysical studies of the structure and function of the flippase will be made possible. New materials and methods developed in the course of this work will also find use in other membrane studies. This work will provide an understanding of the molecular mechanisms controlling membrane asymmetry that may lead to new strategies for the prevention of vascular disease.